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Abstract:

The invention relates to a novel use of the soluble forms of HLA-G in the
treatment or prophylaxis of abnormal B-cell proliferation, such as liquid
cancers of the B type.

Claims:

1. A soluble form of HLA-G suitable as a medicinal product for treatment
or prevention of a B-cell malignant hemopathy.

2. A pharmaceutical composition, comprising: a soluble form of HLA-G; and
at least one pharmaceutically acceptable vehicle, wherein the
pharmaceutical composition is suitable for treatment or prevention of
B-cell malignant hemopathies.

3. The soluble form of HLA-G of claim 1, selected from the group
consisting of HLA-G5, HLA-G6, and HLA-G7.

4. The soluble form of HLA-G of claim 1, in free or monomeric form.

5. The soluble form of HLA-G of claim 1, in the multimeric form.

6. The composition of claim 2, wherein the soluble form of HLA-G is
selected from the group consisting of HLA-G5, HLA-G6, and HLA-G7.

7. The composition of claim 2, wherein the soluble form is in free or
monomeric form.

8. The composition of claim 2, wherein the soluble form is in multimeric
form.

9. The composition of claim 2, wherein the pharmaceutically acceptable
vehicle is suitable for parenteral administration.

10. The composition of claim 2, wherein the pharmaceutically acceptable
vehicle is suitable for administration by inhalation.

11. A product, comprising: a soluble form of HLA-G; and an anticancer
product, as a combined preparation for simultaneous, separate, or
sequential use in treating or preventing a cancer of B-cell malignant
hemopathy.

12. The soluble form of HLA-G of claim 1, in the form of HLA-G5.

13. The composition of claim 2, wherein the soluble form of HLA-G is
HLA-G5.

14. A method of treating a B-cell malignant hemopathy, the method
comprising administering to a subject in need thereof, an effective
amount of the soluble form of HLA-G of claim 1.

15. The method of claim 14, wherein the administering is parenteral.

16. The method of claim 14, wherein the administering is by inhalation.

17. A method of treating a B-cell malignant hemopathy, the method
comprising administering to a subject in need thereof, an effective
amount of the pharmaceutical composition of claim 2.

18. A method of treating a B-cell malignant hemopathy, the method
comprising administering to a subject in need thereof, an effective
amount of the product of claim 11.

19. A method of preparing the pharmaceutical composition of claim 2, the
method comprising combining the soluble form of HLA-G with the at least
one pharmaceutically acceptable vehicle.

20. The method of claim 19, wherein, in the combining, at least one
further anticancer product is combined.

Description:

[0001] The present invention relates to a novel use of the soluble forms
of HLA-G in the treatment of abnormal B-cell proliferation such as liquid
cancers of type B and auto-immune diseases in which the B cells are
activated.

[0002] The antigens of the major histocompatibility complex (MHC) are
divided into several classes, the class I antigens (HLA-A, HLA-B and
HLA-C), which have 3 globular domains (α1, α2 and α3),
the α3 domain being associated with 2 microglobulin, the class II
antigens (HLA-DP, HLA-DQ and HLA-DR) and the class III antigens
(complement).

[0003] The class I antigens comprise, apart from the aforementioned
antigens, other antigens, called nonclassical class I antigens, and
notably the HLA-E, HLA-F and HLA-G antigens.

[0005] The HLA-G gene differs from the other class I genes in that the
codon for translation termination, in phase, is localized at the level of
the second codon of exon 6; in consequence, the cytoplasmic region of the
protein encoded by this HLA-6.0 gene is shorter than that of the
cytoplasmic regions of the HLA-A, -B and -C proteins. Expression of these
isoforms is restricted to a few tissues such as the trophoblast (Kovats
et al., 1990), the thymus (Crisa et al., 1997) and the pancreas (Cirulli
et al., 2006) in nonpathological conditions.

[0006] Other research concerning this nonclassical class I antigen
(ISHITANI et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3947-3951) showed
that the primary transcript of the HLA-G gene can be spliced in several
ways and produces at least 3 different mature mRNAs: the primary
transcript of HLA-G supplies a complete copy (G1) of 1200 bp, a fragment
of 900 by (G2) and a fragment of 600 by (G3). The transcript G1 does not
include exon 7 and corresponds to the sequence described by ELLIS et al.
(mentioned previously), i.e. it codes for a protein that comprises a
signal sequence, three external domains, a transmembrane region and a
cytoplasmic sequence. The mRNA G2 does not include exon 3, i.e. it codes
for a protein in which the α1 and α3 domains are joined
directly; the mRNA G3 contains neither exon 3 nor exon 4, i.e. it codes
for a protein in which the α1 domain and the transmembrane sequence
are joined directly. The splicing that prevails for obtaining the HLA-G2
antigen leads to joining of an adenine (A) (derived from the domain
coding for α1) to a sequence AC (derived from the domain coding for
α3), which leads to the creation of a codon AAC (asparagine) in
place of the codon GAC (aspartic acid), occurring at the start of the
sequence coding for the α3 domain in HLA-G1. The splicing generated
for obtaining HLA-G3 does not lead to formation of a new codon in the
splicing zone.

[0007] The authors of this article also analyzed the various proteins
expressed: the 3 mRNAs are translated into protein in the 0.221-G cell
line. They conclude, without proof, that the HLA-G molecule has a
fundamental role in protection of the fetus against a maternal immune
response (induction of immune tolerance).

[0009] Moreover, some of the inventors have shown the existence of other
spliced forms of HLA-G mRNA: the HLA-G4 transcript, which does not
include exon 4; the HLA-G5 transcript, which includes intron 4, between
exons 4 and 5, thus causing a modification of the reading frame, during
translation of this transcript and in particular the appearance of a stop
codon, after amino acid 21 of intron 4; the HLA-G6 transcript, which
possesses intron 4, but has lost exon 3 (KIRSZENBAUM M. et al., Proc.
Natl. Acad. Sci. USA, 1994, 91, 4209-4213; European Application EP 0 677
582; KIRSZENBAUM M. et al., Human Immunol., 1995, 43, 237-241; MOREAU P.
et al., Human Immunol. 1995, 43, 231-236); and the HLA-G7 transcript,
which includes intron 2, thus causing a modification of the reading
frame, during translation of this transcript and the appearance of a stop
codon after amino acid 2 of intron 2; they also showed that these various
transcripts are expressed in several types of fetal and adult human
cells, notably in T and B lymphocytes (KIRSZENBAUM M. et al., Human
Immunol., 1995, op. cit.; MOREAU P. et al., Human Immunol. 1995, op.
cit.).

[0010] There are therefore at least 7 different HLA-G mRNAs, which
potentially code for 7 isoforms of HLA-G including 4 membrane isoforms
(HLA-G1, G2, G3 and G4) and 3 soluble isoforms (HLA-G5, G6 and G7).

[0011] Preliminary studies have shown that expression of HLA-G molecules
on the surface of target cells obtained by transfection with vectors
comprising the genomic DNA of HLA-G, potentially generating all the
alternative transcripts, makes it possible to protect said target cells
from the lytic activity of the NK cells of the decidual layer of the
maternal endometrium (CHUMBLEY G. et al., Cell Immunol., 1994, 155,
312-322; DENIZ G. et al., J. Immunol., 1994, 152, 4255-4261).

[0012] These preliminary studies were confirmed subsequently; thus, both
the membrane-bound isoforms and the soluble isoforms are immunotolerant:
[0013] they inhibit cytolysis mediated by the NK cells and the CTLs;
[0014] they inhibit the alloproliferative T response. The inhibitory
action of HLA-G on the T cells is described in the literature, including
that emanating from the group of M. CAROSELLA (5-13); [0015] they induce
apoptosis in the T cells and the NK CD8.sup.+ cells.

[0016] Thus, the HLA-G protein exerts its function locally, both when it
is expressed on the surface of the cells and when it is secreted (action
at a distance); it thus provides immune surveillance of the organism
(Teyssier E. et al., Nat. Immunol., 1995, 14, 262-270).

[0017] Its properties of immunotolerance have also been demonstrated in
vitro in many models of tumoral lines transfected with HLA-G (6, 23, 24).
The HLA-G antigens play a key role in establishing and maintaining
immunotolerance by inhibiting the functions of the immunocompetent cells.

[0020] Thus, HLA-G and notably the soluble isoforms such as HLA-G5:
[0021] induce apoptosis of the T CD8+ cells and of the NK cells activated
by binding to the CD8 receptor and stimulation of the Fas/Fas pathway.
[0022] exert their inhibitory effects by a feedback mechanism because
they inhibit the proliferative response of the alloreactive T CD4+ cells
which secrete it. [0023] have immunosuppressive properties, which they
exert via their interaction with the inhibitory receptors variously
expressed on the surface of the various immune cells (NK cells, T cells,
B cells and antigen-presenting cells) (Carosella et al., Trends in
Immunology, 2008, 29, 3, 125-132; patent application FR 2 810 047; Naji
et al., 2007). [0024] also exert their immunosuppressive activity by
effects mediated by cytokines, such as IL-10 and the interferons (patent
application FR 2 810 047).

[0025] The tolerogenic properties of HLA-G have beneficial effects in
disorders of pregnancy, transplantation and auto-immunity and in
inflammatory diseases by limiting the immune reactions, whereas they have
deleterious effects in cancer and after viral infections by permitting
escape of tumor cells or of cells infected by viruses.

[0026] Thus, it is now widely recognized that expression of HLA-G by tumor
cells is a negative factor enabling the latter to inhibit the antitumor
response through interaction of HLA-G with the inhibitory receptors of
type ILT-2 expressed by the T cells and NK cells infiltrating the tumor
(see the special issue of the journal Seminars in Cancer Biology on HLA-G
and cancer (16)). This action of HLA-G therefore leads to tumor
progression and blocking of HLA-G is accordingly now proposed as a new
antitumor therapeutic approach. As an example, we may mention the works
of M. Carosella's team on melanoma, showing the role of HLA-G in
protecting melanomatous cells against the action of the immune system
(17-20). This observation is confirmed in other types of tumors such as
glioma or human renal carcinoma lines protected from an allogenic
cytotoxic response by expression of HLA-G1 and HLA-G5 molecules (10, 21,
22). The many reviews on the role of HLA-G in oncology confirm these
observations (2, 14, 15, 25-27).

[0027] However, all of these works relate to the action of the HLA-G
molecule expressed in solid tumors.

[0028] Surprisingly, the inventors have now shown that the soluble forms
of HLA-G have an antiproliferative action on the B cells of the immune
system. For example, they have shown in particular the inhibitory action
of the soluble forms of HLA-G on the functions of differentiation,
proliferation and antibody secretion of B lymphocytes. These results have
a particularly decisive impact within the scope of B-cell malignant
hemopathies (lymphoma, lymphoid leukemia, myeloma, Burkitt syndrome,
Hodgkin's disease etc.).

[0029] The present invention accordingly relates to the use of a soluble
form of HLA-G for preparing a medicinal product for the treatment or
prevention of B-cell malignant hemopathies, i.e. of pathologies in which
an observed.

[0030] In other words, the present invention relates to the soluble forms
of HLA-G for use as a medicinal product for the treatment or prevention
of B-cell malignant hemopathies.

[0031] Such a use of HLA-G in oncology, in which the B cells are tumoral,
is particularly unexpected, since it runs counter to the present concept
of the role of HLA-G as a mechanism by which tumors evade immune
surveillance (2, 14, 15).

[0032] Now, the inventors have found that HLA-G specifically inhibits the
proliferation of tumor cells of the immune system expressing inhibitory
HLA-G receptors, i.e. principally B cells. HLA-G also inhibits the
proliferation, differentiation to plasmocytes and capacity to secrete
antibodies of abnormally activated B cells, which means they can be used
in auto-immune diseases in which the B cells are abnormally activated.

[0041] As an example, the non-Hodgkin lymphomas are malignant tumors of
the lymphatic system; there are numerous forms, which develop very
differently from one another.

[0042] These lymphomas develop starting from T or B lymphocytes. B-cell
tumors represent 75% of cases in western countries whereas T-cell tumors
are more common in East Asia.

[0043] The incidence of non-Hodgkin lymphomas is increasing throughout the
world; more than 287 000 new cases occur each year, mainly in the
developed countries.

[0044] The non-Hodgkin lymphomas occur more often in the developed
countries (52% of the total number of cases in the world), where their
incidence has increased in the last 20 years, mainly in North America,
Western Europe, Australia, Israel, Saudi Arabia. Lymphoma is also a
tumoral complication observed in 5 to 10% of cases of AIDS.

Abnormally Activated B Cells

[0045] The B cells are said to be abnormally activated when they respond
to auto-antigens.

Soluble Form of HLA-G

[0046] Said soluble form of HLA-G is selected from the group comprising
HLA-G5, HLA-G6 and HLA-G7, preferably HLA-G5. These soluble forms are
well known by a person skilled in the art.

[0047] The use of HLA-G in liquid cancers constitutes an alternative or
complementary treatment, in combination with the treatments usually
employed, as described for example in Keating M. et al. (Hematology,
2003, 153-175); Dighiero G. et al. (The Lancet, 2008, 371, 1017-1029); or
Auer R. et al. (Br. J. Hematol., 2007, 139, 635-644).

[0048] The soluble forms of HLA-G, which have a mechanism of action
radically different from the other anticancer products usually employed,
thus offer, alone or in combination with these other products, a benefit
in cases of (i) poor level of response with the other treatments, (ii)
appearance of resistance to the other treatments and (iii) when the
undesirable effects observed with the other treatments are too great.

[0049] HLA-G5 and, more generally, the soluble form of HLA-G, have, in
liquid cancers, an antiproliferative activity and limit tumor
progression.

[0050] This activity is opposite to that previously described, relating to
solid cancers in which the aim is to block expression of HLA-G, to
eliminate the solid tumor.

[0051] According to the invention, the soluble form of HLA-G employed is:
[0052] either in the free (or monomeric) form, which can optionally form
dimers in solution, [0053] or in multimeric form, notably in aggregated
form on beads, so that the molecule of HLA-G is in the form of multimers,
described as being the functionally optimal conformation of the HLA-G
molecule. In fact, dimers of HLA-G have been described as displaying
greatly increased affinity for the HLA-G receptors compared with the
monomers.

[0054] According to the invention, the abnormal B-cell proliferation is
inhibited both by the soluble form of HLA-G, purified and non-aggregated
on beads, and with the aggregated forms of said soluble form of HLA-G.

[0055] The present invention also relates to a pharmaceutical composition
comprising a soluble form of HLA-G and at least one pharmaceutically
acceptable vehicle for use as a medicinal product for the treatment or
prevention of B-cell malignant hemopathies.

[0056] According to an advantageous embodiment of said composition, said
pharmaceutically acceptable vehicle is suitable for parenteral
administration.

[0057] Administration can be for example intravenous, intramuscular or
subcutaneous.

[0058] According to an advantageous embodiment of said composition, said
pharmaceutically acceptable vehicle is suitable for administration by
inhalation.

[0059] Solutions or suspensions used for subcutaneous application
typically include one or more of the following compounds: a sterile
diluent, such as water, for injectable preparations, a physiological
saline solution, isotonic and buffered, oils, polyethylene glycols,
glycerol, polypropylene glycol or other synthetic solvents; antibacterial
agents such as benzyl alcohol or methylparabens; antioxidants such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates; and agents for adjusting tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases such as hydrochloric
acid or sodium hydroxide.

[0060] These preparations can be in the form of ampules, disposable
syringes or multidose bottles made of glass or plastic.

[0062] For intravenous administration, the preferred vehicles include
physiological saline solutions, bacteriostatic water, Cremophor EL®
(BASF, Parsippany, N.J.) or PBS buffer. In all cases, the composition
must be sterile and fluid. It must be stable in the conditions of
preparation and storage and must comprise preservatives against the
contaminating action of microorganisms such as bacteria or fungi.

[0063] As an example, the vehicle can be a solvent or a dispersion medium
containing for example water, ethanol, a polyol (for example glycerol,
propylene glycol or a liquid polyethylene glycol) and mixtures of these
compounds.

[0064] The correct fluidity can be maintained for example by using
lecithin, or by using surfactants. The action of microorganisms can be
prevented by the administration of various antibacterial and antifungal
agents, for example parabens, chlorobutanol, phenol, ascorbic acid and
thimerosal. Said compositions can also include isotonic agents, for
example sugars or polyalcohols such as mannitol, sorbitol or sodium
chloride.

[0065] Prolonged action of the injectable compositions can be obtained by
adding aluminum monostearate or gelatin to the formulation.

[0066] The present invention also relates to products containing a soluble
form of HLA-G and an anticancer product as combined preparation for
simultaneous, separate or sequential use in the treatment or prevention
of cancers of B-cell malignant hemopathies.

[0067] Apart from the above arrangements, the invention further comprises
other arrangements, which will become clear from the description that now
follows, which refers to examples of application of the method according
to the present invention, as well as to the appended drawings, in which:

[0071]FIG. 8 illustrates inhibition of the differentiation, to malignant
CD138.sup.+ plasmocyte cells, of CD138.sup.- cells from the bone marrow
of patients with multiple myeloma; the cells were sensitized in the
presence (Beads-HLA-G5) or absence (0 and beads) of HLA-G5. Only one of
the 3 repetitions is shown. For the cells sensitized with the culture
medium without beads ("O"): 17 CD138.sup.- cells out of 100
differentiated to CD138+ cells; for the cells sensitized with the culture
medium comprising microbeads alone ("beads"): 18 CD138.sup.- cells out of
100 differentiated to CD138.sup.+ cells; for the cells sensitized with
the culture medium comprising microbeads covered with HLA-G5
(Beads-HLA-G5): 4 CD138 cells out of 100 differentiated to CD138+ cells.

[0072] However, it has to be understood that these examples are given
solely for illustrating the object of the invention, and they do not in
any way constitute a limitation thereof.

[0077] CD138.sup.- cell fractions were supplied by Pasteur-Cerba (Cergy,
France). These cell fractions correspond to mononuclear cells from bone
marrow (BM), isolated from samples obtained from patients with multiple
myeloma, from which the CD138.sup.+ plasmocyte cells had been excluded
using anti-human CD138 magnetic microbeads (Miltenyi Biotech). Informed
consent had been obtained from all the patients in accordance with the
Declaration of Helsinki, and the study was approved by the ethics
committee.

[0082] The protein HLA-G5 and its nucleic acid sequence are described in
patent application EP 0 677 582. The production of a soluble form of an
isoform of HLA-G in a baculovirus is described in detail in Example 1 of
application EP 1 189 627.

[0083] Briefly, the recombinant protein HLA-G5 is produced in SF9 insect
cells, cultivated on TNMFH medium containing 5% of fetal calf serum
(Invitrogen), infected with a baculovirus containing the sequence coding
for HLA-G5 (HLA-G5 baculovirus) or infected with the HLA-G5 baculovirus
as well as with a baculovirus coding for human β2m (Appligene) and
cultivated for 5 days at 27° C. in the presence of 5% CO.

[0084] The apyrogenic protein HLA-G5 is purified from the culture
supernatant of infected SF9 cells by immunoaffinity chromatography with
W6/32 monoclonal antibodies (Sigma-Aldrich).

[0085] Production of Recombinant HLA-G5 Adsorbed on Microbeads

[0086] Briefly, the magnetic microbeads are mono-dispersed particles with
a diameter of 300 nm and are covered with goat antimouse IgG bound
covalently to their surface (Bio-Adembeads goat antimouse, Ademtech).

[0093] The cells are recovered 18 h later and the incorporation of
thymidine in DNA is quantified on a β counter (Wallac 1450,
Pharmacia).

[0094] The peripheral blood mononuclear cells (PBMCs) (105
cells/well) are activated by the mitogen pokeweed (2 μg/ml) in the
absence or in the presence of HLA-G5 beads or of beads alone (2104
beads/cell).

[0095] After 5 days, the cultures are pulsed with 3H-thymidine (1
μCi/well, Amersham, Biosciences). The cells are collected 18 hours
later, and the incorporation of thymidine in DNA is quantified on a
β counter (Wallac 1450, Pharmacia).

[0096] Analysis of the Cell Cycle

[0097] The Raji cells are treated either with the HLA-G5 beads or with the
beads alone (5104 beads/cell). After 24 h, the Raji cells are fixed
in ethanol at 70% (v/v) in PBS buffer and incubated overnight at
4° C. After washing, the cells are incubated in PBS buffer
containing 40 μg/ml of propidium iodide (Sigma) and 100 μg/ml of
DNase without RNase A in ice, for at least 10 min.

[0098] The parameters of the cell cycle are obtained using an LSR® flow
cytometer and the software Cell Quest® (Becton Dickinson).

[0099] The distribution of the cell cycle is determined by automatic
analysis employing the software ModFit LT® with AutoDebris® and
AutoAggregates®. The percentage of cells in each phase of the cell
cycle (G0/G1, S and G2) is calculated as described in Menier C. et al.
(Leukemia, 2008, 22, 578-584).

[0101] The PBMCs (106 cells/ml) are activated by the mitogen pokeweed
(2 μg/ml) in the absence or in the presence of HLA-G5 beads or of
beads alone (2103 beads/cell).

[0102] After 5 days, the cells are harvested from the cultures by the
cytospin technique (superfrost/plus plates (Merck, Strasbourg)) and
Cytospin 3.sup.- (Shandon).

[0103] For staining, the cells are fixed in ethanol and incubated for 30
min with goat anti-IgG, anti-IgA and anti-IgM human antibodies labeled
with FITC (fluorescein 5-isothiocyanate) or with control antibodies
labeled with FITC.

[0104] The nuclei are labeled red with propidium iodide.

[0105] The plates are analyzed using a fluorescence microscope (Biorad MRC
1024). The percentage of plasmocytes positive for intracytoplasmic Ig is
established by counting the cells with fluorescent cytoplasm.

[0106] ELISA

[0107] The PBMCs (106 cells/ml) are activated by the mitogen pokeweed
(2 μg/ml) in the absence or in the presence of HLA-G5 beads or of
beads alone (2103 beads/cell). The human IgA and IgG secreted in the
supernatants from culture of PBMCs are measured by means of the IgG ELISA
and IgA ELISA quantification kits (Bethyl, Montgomery, Tex.), according
to the manufacturer's instructions.

[0108] Tests of Cellular Differentiation

[0109] The CD138.sup.- cell fractions obtained from bone marrow samples
from patients with multiple myeloma were sensitized in vitro, for 18 to
24 h, with culture medium containing either microbeads covered with
HLA-G5, or microbeads alone, or culture medium not containing microbeads.
The cells were then recovered (without the microbeads) and cultivated for
21 days.

[0110] After the 3 weeks of culture, expression of CD138 on the surface of
the cells among the population of CD45.sup.+ cells was determined by flow
cytometry.

[0111] Flow Cytometry

[0112] The antibodies used for the analyses by flow cytometry were
conjugated with FITC, PE (Phycoerythrin), DPE (dinitrophenyl) or PC5
(Phycoerythrin-cyanin 5) (Beckman Coulter and BD Pharmingen). Briefly,
the cells were incubated for 30 min at 4° C. in 20% of human serum
and then labeled with the antibodies. Isotype control was used regularly
for evaluating and compensating the nonspecific signal. The cells were
analyzed on an EPIC XL4 flow cytometer using the Expo32 software (Beckman
Coulter).

[0113] Statistical Analysis

[0114] All the data are representative of experiments conducted at least
three times. The significance was evaluated by the unpaired t test,
regarding p<0.05 as significant.

EXAMPLE 2

Inhibition of Proliferation of B Tumor Cells (B Raji Tumoral Line)

[0115] The Raji tumoral cell line described in Example 1 was treated with
beads alone or with beads covered with the soluble form HLA-G5. After 24
hours, the proliferation of the B Raji tumor cells was analyzed by
incorporation of tritiated thymidine. The percentage inhibition of
proliferation is shown in FIG. 1 and corresponds to the mean value
obtained in four experiments.

[0116] These results are illustrated in FIG. 1 with the B Raji tumoral
line, derived from a patient with Burkitt syndrome, for which there is a
significant, dose-dependent decrease in proliferation after treatment
with the soluble form HLA-G5. This inhibition of proliferation of B Raji
tumor cells by HLA-G5 passes through a stoppage in phase G1 of the cell
cycle (Table I).

[0117] The distribution during the cell cycle was defined by labeling the
Raji cells with propidium iodide after 24 h of a treatment with
beads-HLA-G5 or with beads alone at a rate of 50 000 beads/cell, which
corresponds to 50 ng/ml of HLA-G5. The results are represented as the
percentage of cells in each phase of the cycle.

[0118] Results similar to those of Example 2 were obtained with another
Burkitt B tumoral line, the Daudi line as well as with lines derived from
another type of lymphoproliferation, namely myelomas.

[0119] The Daudi, OPM-2 and RPMI 8226 cell lines described in Example 1
were treated with beads alone or with beads covered with the soluble form
HLA-G5 or were not treated. After 24 hours, the proliferation of these B
tumor cells was analyzed by incorporation of tritiated thymidine. The
number of tumor cells introduced in the test varies from 10 000 to 30 000
cells per well. The number of beads is fixed and is 50 000 beads per
cell.

[0120] The beads covered with the protein HLA-G5 inhibit the proliferation
of these B tumor cells in all cases (FIGS. 5, 6 and 7).

[0121] This experiment is representative of three independent experiments.

[0123] Peripheral blood mononuclear cells (PBMCs) isolated from a blood
sample from healthy individuals were stimulated by the mitogenic agent
pokeweed mitogen (PWM) in the presence (Beads-HLA-G5) or in the absence
(Beads) of beads covered with the soluble form HLA-G5. After 5 days, the
proliferation of the stimulated B cells was analyzed by incorporation of
tritiated thymidine. These results represent the mean value obtained in 6
independent experiments. Inhibition of proliferation connected with the
treatment with HLA-G5 is statistically significant. These results are
shown in FIG. 2.

[0125] Peripheral blood mononuclear cells (PBMCs) isolated from a blood
sample from healthy individuals were stimulated by the mitogenic agent
pokeweed mitogen (PWM) in the presence (Beads-HLA-G5) or in the absence
(Beads) of beads covered with the soluble form HLA-G5. After 5 days, the
percentage of B cells differentiated to plasmocytes with intracytoplasmic
immunoglobulins (IgIC) was determined by immunofluorescence. Inhibition
of differentiation connected with the treatment with HLA-G5 is
statistically significant. These results are shown in FIG. 3.

[0126] Inhibitory Action of HLA-G5 on the Capacity of B Cells to Secrete
Antibodies (FIG. 4)

[0127] Peripheral blood mononuclear cells (PBMCs) isolated from a blood
sample from healthy individuals were stimulated by the mitogenic agent
pokeweed mitogen (PWM) in the presence (Beads-HLA-G5) or in the absence
(Beads) of beads covered with the soluble form HLA-G5. After 5 days, the
level of immunoglobulins IgA and IgG in the culture supernatants was
measured by the immunoenzyme technique. Inhibition of the secretion of
antibodies connected with the treatment with HLA-G5 is statistically
significant. These results are shown in FIG. 4.

EXAMPLE 5

Inhibition Ex Vivo, by HLA-G5, of the Differentiation of CD138 Cells from
the Bone Marrow of Patients with Multiple Myeloma to Malignant Plasma
Cells CD138.sup.+

[0128] CD138.sup.+ cell tractions obtained from bone marrow from patients
with multiple myeloma were sensitized for 18 h at 24 h with culture
medium containing either microbeads covered with HLA-G5, or microbeads
alone, or with culture medium not containing microbeads. After 3 weeks of
culture without microbeads, the differentiation of CD138.sup.- cells to
CD138+ cells was analyzed by flow cytometry.

[0129] The results are shown in FIG. 8, from which it can be seen that
HLA-G5 inhibits, at a level of 68% ((17×4)/100), the capacity of
the CD138.sup.- progenitor cells to differentiate to CD138.sup.+ cancer
cells. In contrast, in the absence of HLA-G5 (O and beads), a
significantly larger number of CD138.sup.- progenitor cells differentiate
to CD138.sup.+ malignant plasmocyte cells (respectively 17/100 and
18/100).